Although the principle of a flying spot scanner has been known for many years, its application in microscopy has prospered only in the last few years as the necessary technology has been developed. Stable laser light sources and fast electronic image acquisition and storage technology are necessary ingredients for a scanning microscope. While the imaging properties of a non-confocal scanning microscope are very similar to those of conventional microscopes, a new domain is opened by confocal scanning microscopes. The resolution provided by such devices is only moderately increased, but the vastly improved depth discrimination they provide allows the generation of three dimensional images without complicated deconvolution algorithms. The depth discrimination reduces background, and this, together with the use of a single high quality detector such as a photomultiplier, allows quantitative studies with high spatial resolution.
The resolution along the optical axis of a confocal scanning microscope provides useful discrimination against background scattering or fluorescence arising above and below the plane of focus in a transparent object. It is also very helpful in constructing three dimensional fluorescent images from a series of sections and for the use of quantitative fluorescence indicators or for mapping of fluorescent markers of cell surface receptors on non-planar surfaces. Such devices provide slightly better lateral resolution, much better depth field discrimination, and orders of magnitude better background discrimination under ideal conditions than was available with prior devices, under ideal conditions.
Scanning can be carried out either by moving the specimen stage under a stationary beam or by precisely synchronized optical scanning of both the illumination and the fluorescent response signals. Although the moving stage solution is preferable from an optical point of view, it puts limits on sample access and mounting, the use of environmental chambers, and electrical recording with microelectrodes. Accordingly, the moving spot approach is often favored. Such a moving spot may be produced by the use of mirrors mounted on galvonometer scanners, although this limits the obtainable frame frequency. The use of accousto-optical deflectors interferes with the confocal spatial filtering in fluorescence microscopy because of their strong dispersion. Although polygonal mirrors are faster than galvonometer scanners, one alone does not allow a vector mode of operation.
A conventional arc light source can be used for many applications of a confocal scanning microscope which utilizes a rotating disc illuminator, but apparently inescapable intensity modulations limit its use for quantitative applications. In such devices, the image is formed either through a dual set of confocal pin holes in the disc, or, in recent versions, through the illumination pinholes themselves.
Confocal scanning microscopes in which a single point illuminated by a laser is scanned across the moving object work quite well at slow scanning speeds, and good laser scanning micrographs have been obtained using fluorescence markers that absorb and emit visible light. However, confocal scanning images with fluorophores and fluorescent chemical indicators that are excited by the ultraviolet part of the spectrum have not been available, largely because of the lack of suitable microscope lenses, which must be chromatically corrected and transparent for both absorption and emission wavelengths, but also because of the damage done to living cells by ultraviolet light. Furthermore, the limitations of ultraviolet lasers have inhibited such usage.
Fluorescence microscopy is further limited, in all of its manifestations, by the photobleaching of fluorophores in the target material, for the exciting light slowly photobleaches the fluorophores while it is exciting fluorescence. Even in laser scanning confocal fluorescence microscopy, essentially the same photobleaching is incurred as happens in wide field microscopy, because the focused exciting light still illuminates the full depth of the target specimen uniformly, in a time average, as it scans the plane of focus. Photobleaching is particularly troublesome in a three-dimensional image reconstruction because many two-dimensional images are required for this purpose, and the acquisition of each two-dimensional image produces photobleaching throughout the specimen.